Structural Requirements for the Ubiquitin-associated Domain of the mRNA Export Factor Mex67 to Bind Its Specific Targets, the Transcription Elongation THO Complex Component Hpr1 and Nucleoporin FXFG Repeats

Abstract

The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (nucleoporin) FG repeats and Hpr1, a component of the TREX⅐THO complex that functions to link transcription and export. Using fluorescence resonance energy transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin peptide and define residues engaged in the interaction and those involved in the FXFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FXFG nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FXFG is unable to bind mono-or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.Transcripts generated by RNA polymerase II undergo a carefully orchestrated series of processing steps, including 5Ј capping, splicing, 3Ј end cleavage, and polyadenylation before being exported to the cytoplasm. The steps of mRNA biogenesis leading to export-competent ribonucleoprotein particles (mRNPs) 5 are intimately coupled (1, 2). This coordination of mRNA biogenesis is mediated by a diverse range of RNA-binding proteins and maturation enzymes (reviewed in Refs. 2 and 3). Moreover, during processing, the transcripts are under the constant surveillance of RNA quality control mechanisms that ensure that only correctly processed mRNAs are exported to the cytoplasm for translation (4 -7). From yeast to human, translocation of fully mature mRNPs to the cytoplasm through nuclear pore complex is thought to be mediated primarily by Mex67/NXF1 (also known as TAP), the primary mRNA export receptor, which forms a heterodimer with Mtr2/NXT1 (P15) that interacts directly with nuclear pore complex proteins (FG nucleoporins) that have characteristic FG sequence repeats (8 -10). Previous work has shown that, like other nuclear transport factors (reviewed in Refs. 11 and 12), the Mex67/TAP UBA domain binds specifically to the two Phe rings of the FXFG nucleoporin-binding motif (13,14). Because Mex67/NXF1 has low intrinsic affinity for mRNAs, it is recruited to the mRNP by several RNA-binding adaptors, including members of the THO⅐TREX complex that couples transcription elongation to mRNA export (2,15,16) as well as the TREX-2 complex, Yra1/ ALY and Sub2/UAP56 (reviewed in Ref. 3).Recent studies have shown that the ubiquitin pathway participates in the regulation of several key cellular functions including mRNA nuclear export (17)(18)(19). Ubiquitin is a small 76-residue protein that can be covalently linked as a mono...

Nuclear export of mRNA and its regulation by ubiquitylation

“…Further, Mex67 interacts with Hpr1 of the THO complex via its ubiquitin-associated (UBA) domain. [51][52][53] Mex67 thus recruited to mRNA interacts with FG-repeats of nucleoporins, and hence, transports mRNA across NPC. In the cytoplasmic side of the NPC, Mex67 is dissociated from mRNA with the help of Dbp5 (a DEAD box RNA helicase with ATPase and RNA unwinding activities), InsP6 (inositol hexaphosphate) and Gle1, and then, recycled back to the nucleus for subsequent rounds of mRNA export.…”

“…Although several studies [10][11][12][51][52][53]75 have implicated the HECT domain containing E3 ligases such as Tom1 and Rsp5 in regulation of mRNA export, there could be additional E3 ligase(s) with the HECT domain or SCF (Skp1-Cullin-F-Box protein) ubiquitin ligase(s) with the RING domain that may control mRNA export via ubiquitylation of some specific mRNA export-associated factor(s). In fact, our recent work has implicated an F-box protein, Mdm30, in stimulation of the nuclear export of mRNA.…”

“…Such protection is mediated by the recognition of the ubiquitinatedHpr1 by the UBA domain of Mex67. [51][52][53] Thus, the stability of Hpr1 may have some role in mRNA export. Indeed, the deletion of HPR1 or the stabilization of Hpr1 by inactivation of Rsp5 is correlated with the defect in nuclear export of mRNA.…”

An E2 Accessory Domain Increases Affinity for the Anaphase-promoting Complex and Ensures E2 Competition

“…Several previously characterized UBA domains can bind both ubiquitin and another partner, sometimes simultaneously (27)(28)(29). Because the affinity of the UBA domain for a single ubiquitin is low (ϳ230 M) but is considerably higher for a ubiquitin chain (ϳ37 nM for Lys-48-linked tetraubiquitin), it is possible that as the ubiquitin chain on substrates grows longer it interacts with the UBA domain.…”

“…However, previous studies indicate that the UBA domain does not bind the ubiquitin covalently linked to the UBC domain (the donor ubiquitin) (26) or the ubiquitin attacking the E2-ubiquitin conjugate (the acceptor ubiquitin) (3). UBA domains in other proteins have been shown to bind non-ubiquitin folds (27)(28)(29). It therefore remains unclear how a putative ubiquitin-binding domain promotes a productive interaction between Ubc1 and the APC/C.…”